DESCRIPTION (Applicant's Abstract): Mistakes in chromosome segregation during meiosis or mitosis can lead to spontaneous abortion or to abnormalities such as Down syndrome, They have also been implicated in the genetic progressions leading to cancer and aging. Our laboratory studies several genes in Drosophila that are required for proper chromosome segregation. We believe that this organism offers important advantages in genetics, genomics and cytology that provide unique opportunities to investigate chromosome behavior and cell cycle progression during cell division. We first focused on an evolutionarily conserved gene called zwlO. Mutations in zwlO not only disrupt chromosome segregation, but they also prevent the operation of the spindle assembly checkpoint that regulates anaphase onset. The ZW1O protein displays an unusual, dynamic pattern of localization during the cell cycle, moving between chromosomal kinetochores and kinetochore microtubules. We found that this pattern is influenced by bipolar tension across individual chromosomes. We also discovered that ZW1O is part of a large protein complex, one of whose activities is to target the molecular motor dynein to the kinetochore. The first specific aim describes further investigations on ZW1O.We will use real-time video microscopy to characterize the distribution of a ZW1O-GFP fusion protein during the cell cycle. We will determine which domains of ZW1O are required for various aspects of its intracellular distribution and function. We will analyze double mutant combinations to position ZW1O in the biochemical pathways underlying the spindle checkpoint and other aspects of anaphase onset. We will also test whether all aspects of the zw10 mutant phenotype, including its effects on the spindle checkpoint, are caused by the absence of dynein at the kinetochore. The second specific aim is to characterize biochemically and genetically the large complex of which ZW1O is a part. Preliminary efforts to purify the complex by affinity chromatography have been promising. We will use several techniques to verify the association of candidate proteins with ZW1O. We will then identify these proteins by mass spectrometry, and will determine whether they are subject to post-translational modification. Our ultimate goal is to obtain antibodies against these proteins, as well as mutations in the genes encoding them. The third specific aim will exploit our identification in the previous funding period of a set of new mutations that cause precocious sister chromatid separation, aneuploidy, or metaphase arrest in Drosophila. We will study the phenotypes associated with these mutations in more detail, clone selected mutant genes, and then investigate the intracellular location of the corresponding gene products. Because we are focusing on genes not previously known to function in any aspect of mitosis, we believe the genetic and antibody reagents obtained by the proposed studies will provide a broadened and unique view of the events occurring at anaphase onset.